ABSTRACTActive segregation of Escherichia coli low-copy-number plasmid R1 involves formation of a bipolar spindle made of left-handed double-helical actin-like ParM filaments. ParR links the filaments with centromeric parC plasmid DNA, while facilitating the addition of subunits to ParM filaments. Growing ParMRC spindles push sister plasmids to the cell poles. Here, using modern electron cryomicroscopy methods, we investigate the structures and arrangements of ParM filaments in vitro and in cells, revealing at near-atomic resolution how subunits and filaments come together to produce the simplest known mitotic machinery. To understand the mechanism of dynamic instability, we determine structures of ParM filaments in different nucleotide states. The structure of filaments bound to the ATP analogue AMPPNP is determined at 4.3 Å resolution and refined. The ParM filament structure shows strong longitudinal interfaces and weaker lateral interactions. Also using electron cryomicroscopy, we reconstruct ParM doublets forming antiparallel spindles. Finally, with whole-cell electron cryotomography, we show that doublets are abundant in bacterial cells containing low-copy-number plasmids with the ParMRC locus, leading to an asynchronous model of R1 plasmid segregation.

Figure 10: Validation of the doublet model(a) Two ParM monomers arranged in an anti-parallel orientation, as obtained from the ParM cryo-EM doublet model. (b) Two ParM monomers arranged in an anti-parallel orientation, obtained from crystal packing of a monomeric ParM X-ray structure (PDB 4A62) 3. (c) Two residues at the interface of the doublet (see ED Table 2), S19 and G21 were mutated to arginine to improve affinity of ParM filaments to each other. Cryo-EM images of the mutant protein with AMPPNP show spontaneous doublet formation and filament bundling without crowding agent, validating the doublet model. This experiment was repeated six times.

Mentions:
Using the class averages and the directionality assignment, we obtained an averaged model for the ParM doublet (Fig. 3d-g, ED Fig. 5e-f, Video 4). Two ParM monomers from adjoining filaments in the doublet model were found to be in a similar orientation as observed in a previous crystal structure of ParM (ED Fig. 6a-b)3. The model of the doublet predicts residues in ParM that should be important in doublet formation (Fig. 3f-g, ED Table 2) and confirmed earlier work, including mutations that modulate the strength of the inter-filament contact. One such set of mutations consisted of S19R and G21R 3. These mutations had been selected previously based on the fact that they are located the furthest away from the filament axis, essentially sticking out, but are shown here directly to be involved in the inter-filament contact. In line with this, mutant ParM(S19R, G21R) spontaneously formed doublets and bundles (ED Fig. 6c), without any crowding agent present in solution, validating both the previous Total Internal Reflection Fluorescence (TIRF) data 3 as well as the current atomic model of the ParM doublet.

Figure 10: Validation of the doublet model(a) Two ParM monomers arranged in an anti-parallel orientation, as obtained from the ParM cryo-EM doublet model. (b) Two ParM monomers arranged in an anti-parallel orientation, obtained from crystal packing of a monomeric ParM X-ray structure (PDB 4A62) 3. (c) Two residues at the interface of the doublet (see ED Table 2), S19 and G21 were mutated to arginine to improve affinity of ParM filaments to each other. Cryo-EM images of the mutant protein with AMPPNP show spontaneous doublet formation and filament bundling without crowding agent, validating the doublet model. This experiment was repeated six times.

Mentions:
Using the class averages and the directionality assignment, we obtained an averaged model for the ParM doublet (Fig. 3d-g, ED Fig. 5e-f, Video 4). Two ParM monomers from adjoining filaments in the doublet model were found to be in a similar orientation as observed in a previous crystal structure of ParM (ED Fig. 6a-b)3. The model of the doublet predicts residues in ParM that should be important in doublet formation (Fig. 3f-g, ED Table 2) and confirmed earlier work, including mutations that modulate the strength of the inter-filament contact. One such set of mutations consisted of S19R and G21R 3. These mutations had been selected previously based on the fact that they are located the furthest away from the filament axis, essentially sticking out, but are shown here directly to be involved in the inter-filament contact. In line with this, mutant ParM(S19R, G21R) spontaneously formed doublets and bundles (ED Fig. 6c), without any crowding agent present in solution, validating both the previous Total Internal Reflection Fluorescence (TIRF) data 3 as well as the current atomic model of the ParM doublet.

ABSTRACTActive segregation of Escherichia coli low-copy-number plasmid R1 involves formation of a bipolar spindle made of left-handed double-helical actin-like ParM filaments. ParR links the filaments with centromeric parC plasmid DNA, while facilitating the addition of subunits to ParM filaments. Growing ParMRC spindles push sister plasmids to the cell poles. Here, using modern electron cryomicroscopy methods, we investigate the structures and arrangements of ParM filaments in vitro and in cells, revealing at near-atomic resolution how subunits and filaments come together to produce the simplest known mitotic machinery. To understand the mechanism of dynamic instability, we determine structures of ParM filaments in different nucleotide states. The structure of filaments bound to the ATP analogue AMPPNP is determined at 4.3 Å resolution and refined. The ParM filament structure shows strong longitudinal interfaces and weaker lateral interactions. Also using electron cryomicroscopy, we reconstruct ParM doublets forming antiparallel spindles. Finally, with whole-cell electron cryotomography, we show that doublets are abundant in bacterial cells containing low-copy-number plasmids with the ParMRC locus, leading to an asynchronous model of R1 plasmid segregation.